Brassinosteroids
Brassinosteroids (BRs) are a group of naturally occurring steroidal plant hormones that are required for plant growth and development. The first identified BR, Brassinolide, was discovered in 1973, when it was shown that pollen extract from Brassica napus could promote stem elongation and cell division. There are few reports on the physiological effects of brassinosteroids in the growth and development of rice and other plants of the Gramineae family. Physiological research indicates that exogenous brassinosteroids alone, or in combination with auxin, enhance bending of the lamina joint in rice. The total yield of Brassinosteroids from 230 kg of Brassica napus pollen, however, was only 10 mg. Extract from the plant Lychnis viscaria contains a relatively high amount of BRs. Lychnis viscaria is said to increase the disease resistance of surrounding plants. In Germany, extract from the plant is allowed for use as a “plant strengthening substance.” Since their initial discovery, over seventy BR compounds have been isolated from plants.
BRs have been shown to be involved in numerous plant processes: promotion of cell expansion and cell elongation (working with auxin); cell division and cell wall regeneration (the mechanism of which is still to be determined); promotion of vascular differentiation; pollen elongation for pollen tube formation; acceleration of senescence in dying tissue cultured cells; and providing protection during chilling and drought stress.
Treatment with low or high concentrations of brassinosteroids promotes or inhibits the growth of roots in rice, respectively (Radiet al. J. Crop Sci. 57, 191 198 (1988)). Brassinosteroids also promote the germination of rice seeds (Yamaguchi et al. Stimulation of germination in aged rice seeds by pre-treatment with brassinolide, in Proceeding of the fourteenth annual plant growth regulator society of America Meeting Honolulu, ed. Cooke A R), pp. 26 27 (1987)). The lamina joint of rice has been used for a sensitive bioassay of brassinosteroids (Maeda Physiol. Plant. 18, 813 827 (1965); Wada et al. Plant and Cell Physiol. 22, 323 325 (1981); Takeno et al. Plant Cell Physiol. 23, 1275 1281 (1982)), because of high sensitivity thereof to brassinosteroids. In etiolated wheat seedlings treatment with brassinolide or its derivative, castasterone, stimulates unrolling of the leaf blades (Wada et al. Agric. Biol. Chem. 49, 2249 2251 (1985)).
Brassinosteroids are recognized as a class of plant hormones through the combination of molecular genetics and researches on biosyntheses (Yokota Trends in Plant Sci., 2, 137 143 (1997)). Most of the C28-brassinosteroids are common vegetable sterols, and they are considered to be biosynthesized from campesterol, which has the same carbon side chain as that of brassinolide. The basic structure of BR is presented below.

Although the sites for BR synthesis in plants have not, to date, been experimentally demonstrated, one well-supported hypothesis is that as BR biosynthetic and signal transduction genes are expressed in a wide range of plant organs, all tissues produce BRs. Since the chemistry of brassinosteroids was established, biological activities of these homologues have been extensively studied, and their notable actions on plant growth have been revealed, which include elongation of stalks, growth of pollen tubes, inclination of leaves, opening of leaves, suppression of roots, activation of proton pump (Mananda, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 23 52 (1988)), acceleration of ethylene production (Schlagnhaufer et al., Physiol. Plant 61, 555 558 (1984)), differentiation of vessel elements (Iwasaki et al., Plant Cell Physiol., 32, pp. 1007 1014 (1991); Yamamoto et al. Plant Cell Physiol., 38, 980 983 (1997)), and cell extension (Azpiroz et al. Plant Cell, 10, 219 230 (1998)). Furthermore, mechanisms and regulations of physiological actions of brassinosteroids have been revealed by a variety of studies on their biosynthesis (Clouse, Plant J. 10, 18 (1996); Fujioka et al. Physiol. Plant 100, 710 715 (1997)).
Brassionsteroid Signaling
In the 1990s, it was discovered in Arabidopsis that BRs are essential plant hormones through analysis of mutant plants unable to naturally synthesize BRs. These Arabidopsis mutants which show characteristic dwarfism, e.g. dwf1: Feldman et al. Science 243, 1351 1354 (1989); dim: Takahashi et al. Genes Dev. 9, 97 107 (1995); and cbb1: Kauschmann et al. Plant J. 9, 701 703 (1996) and their corresponding structural photomorphogenesis and dwarfism are known (e.g. cpd: Szekeres et al. Cell, 85, 171 182 (1997)) and de-etiolation (det2: Li et al., Science 272, 398 401 (1996); Fujioka et al. Plant Cell 9, 1951 1962 (1997)). The morphologic changes are directly related to their deficiency in BR biosynthesis. BRs are also essential in other plants, as demonstrated with studies on a dwarf mutant of Pisum sativum (Nomura et al. Plant Physiol. 113, 31 37, 1997). In all these mutant plants, use of brassinolide will negate the severe dwarfism.
The mechanism by which BR can propagate its effects starts with a cell receptor to interact with a BR. Receptors may be located on the surface of a cell, or within the cell itself Cell-surface receptor kinases activate cellular signal transduction pathways upon perception of extracellular signals, thereby mediating cellular responses to the environment and to other cells. The Arabidopsis genome encodes over 400 receptor-like kinases (RLKs) (Shiu et al., Plant Cell 16, 1220 (May, 2004)). Some of these RLKs function in growth regulation and plant responses to hormonal and environmental signals. However, the molecular mechanism of RLK signaling to immediate downstream components remains poorly understood, as no RLK substrate that mediates signal transduction has been established in Arabidopsis (Johnson et al., Curr Opin Plant Biol 8, 648 (December, 2005)).
The use of Brassionsteroid-insensitive Arabidopsis mutants allowed for the identification of several components of Brassinosteroid signal transduction, including the leucine-rich-repeat (LRR) receptor-like kinases (RLK), brassinosteroid-insensitive 1 (BRI1) and BRI1-associated receptor-kinase (BAK1), the glycogen synthase kinase 3 (GSK3)-like kinase brassinosteroid-insensitive 2 (BIN2), the phosphatase bri1 suppressor 1 (BSU1), and two transcription factors brassinazole-resistant 1 (BZR1) and brassinazole resistant 2 (BZR2)/bri1-EMS-suppressor 1 (BES1). Meanwhile, it has been reported that genetic regulation of the brassinosteroid metabolism makes plants highly sensitive to brassinosteroids, and thus an effect of brassinosteroid administration is markedly enhanced (Neff et al. Proc. Natl. Acad. Sci., USA 96, 15316 23 (1999)).
Brassinosteroids bind to the extracellular domain of the receptor kinase BRI1 to activate a signal transduction cascade that regulates nuclear gene expression and plant development. Many components of the brassinosteroid signaling pathway have been identified and studied in detail. However, the substrate of BRI1 kinase that transduces the signal to downstream components remains unknown.
BRI1 is an RLK that functions as the major receptor for the steroid hormones brassinosteroids (Johnson et al., Curr Opin Plant Biol 8, 648 (December, 2005)). Brassinosteroids bind the extracellular domain of BRI1 to activate its kinase activity, initiating a signal transduction cascade that regulates nuclear gene expression and a wide range of developmental and physiological processes (FIG. 5) (Vert et al., Annu Rev Cell Dev Biol 21, 177 (2005)). Many components of the BR signaling pathway have been identified and much detail has been revealed about how BR activates BRI1 (Wang et al., Nature 410, 380 (Mar. 15, 2001); Kinoshita et al., Nature 433, 167 (Jan. 13, 2005); Wang et al., Plant Cell 17, 1685 (June, 2005); Wang et al., Science 313, 1118 (Aug. 25, 2006); Wang et al., Dev Cell 8, 855 (June, 2005)) and how phosphorylation by downstream GSK3-like kinase, BIN2, regulates the activity of the nuclear transcription factors that mediate BR-responsive gene expression (FIG. 5) (Vert et al., Annu Rev Cell Dev Biol 21, 177 (2005); Wang et al., Dev Cell 2, 505 (April, 2002); He et al., Science 307, 1634 (2005); Yin et al., Cell 120, 249 (Jan. 28, 2005); Vert et al., Nature 441, 96 (May 4, 2006); Gampala et al., Dev Cell 13, 177 (August, 2007)). However, no direct interaction has been observed between BRI1 and BIN2, and it remains unclear how BRI1 kinase at the plasma membrane transduce the signal to cytoplasmic components of the BR pathway (Gendron et al., Curr Opin Plant Biol 10, 436 (October 2007)). Thus, there is a need to identify the kinases that transduce the signal from the BRI1 receptor to BIN2.